Methods in molecular biology vol 1600 microbial toxins methods and protocols

Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protei

Microbial Toxins

Otto Holst Editor

Methods and Protocols

Second Edition

Methods in

Molecular Biology 1600

Me t h o d s i n Mo l e c u l a r Bi o l o g y

Series Editor

John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

Research Center Borstel, Leibniz-Center for Medicine and Biosciences,

Borstel, Schleswig-Holstein, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

ISBN 978-1-4939-6956-2 ISBN 978-1-4939-6958-6 (eBook)

DOI 10.1007/978-1-4939-6958-6

Library of Congress Control Number: 2017937053

© Springer Science+Business Media LLC 2017

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction

on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to

be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

The registered company is Springer Science+Business Media LLC

The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Editor

Otto Holst

Research Center Borstel

Leibniz-Center for Medicine and Biosciences

Borstel, Schleswig-Holstein, Germany

In the year 2000, a first methods collection entitled Bacterial Toxins: Methods and Protocols

was published which contained 20 chapters on protein toxins and endotoxin from bacteria

and cyanobacteria Then, in 2011, a next such collection was published, entitled Microbial Toxins: Methods and Protocols, which included both, protocols on (cyano)bacterial and mold

fungus toxins, with some focus on aflatoxins In both cases, the idea was to support ers of various scientific disciplines with detailed descriptions of state-of-the-art protocols and, since the books turned out to be quite successful, it is quite obvious that this aim could

research-be achieved Based on this success, a second volume entitled Microbial Toxins: Methods and Protocols is presented now which contains protocols on (cyano)bacterial and mold fungus

toxins, with a rather strong focus on Gram-negative endotoxins (lipopolysaccharides).The interest of researchers across a broad spectrum of scientific disciplines in the field

of microbial toxins is clearly unbroken As many other fields do, this field makes use of a broad variety of biological, chemical, physical, and medical approaches, and researchers dealing with any microbial toxin should be familiar with various techniques from all these

disciplines It is our hope that the book Microbial Toxins: Methods and Protocols, Second Edition can strongly support researchers here.

Microbial Toxins: Methods and Protocols, Second Edition comprises 17 chapters

present-ing state-of-the- art techniques that are described by authors who have regularly been uspresent-ing the protocol in their own laboratories Each chapter begins with a brief introduction to the method which is followed by a step-by-step description of the particular method Also, and importantly, all chapters possess a Notes section in which e.g difficulties, modifications and limitations of the techniques are exemplified Taken together, our volume should prove useful to many scientists, including those without any previous experience with a particular technique

Preface

Contents

Preface v Contributors ix

1 Detection of Cholera Toxin by an Immunochromatographic Test Strip 1

Eiki Yamasaki, Ryuta Sakamoto, Takashi Matsumoto, Biswajit Maiti,

Kayo Okumura, Fumiki Morimatsu, G Balakrish Nair,

and Hisao Kurazono

2 Electrochemical Aptamer Scaffold Biosensors for Detection

of Botulism and Ricin Proteins 9

Jessica Daniel, Lisa Fetter, Susan Jett, Teisha J Rowland,

and Andrew J Bonham

3 A Cell-Based Fluorescent Assay to Detect the Activity of AB Toxins

that Inhibit Protein Synthesis 25

Patrick Cherubin, Beatriz Quiñones, Salem Elkahoui, Wallace Yokoyama,

and Ken Teter

4 Molecular Methods for Identification of Clostridium tetani

by Targeting Neurotoxin 37

Basavraj Nagoba, Mahesh Dharne, and Kushal N Gohil

5 Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food 49

Alexandra Morlay, Agnès Roux, Vincent Templier, Félix Piat,

and Yoann Roupioz

6 Aptamer-Based Trapping: Enrichment of Bacillus cereus Spores

for Real-Time PCR Detection 61

Christin Fischer and Markus Fischer

7 Detection of Yersinia pestis in Complex Matrices by Intact Cell

Immunocapture and Targeted Mass Spectrometry 69

Jérôme Chenau, François Fenaille, Stéphanie Simon, Sofia Filali,

Hervé Volland, Christophe Junot, Elisabeth Carniel,

and François Becher

8 A Method to Prepare Magnetic Nanosilicate Platelets for Effective

Removal of Microcystis aeruginosa and Microcystin-LR 85

Shu-Chi Chang, Bo-Li Lu, Jiang-Jen Lin, Yen-Hsien Li,

and Maw-Rong Lee

9 An Immunochromatographic Test Strip to Detect Ochratoxin A

and Zearalenone Simultaneously 95

Xiaofei Hu and Gaiping Zhang

10 Endotoxin Removal from Escherichia coli Bacterial Lysate

Using a Biphasic Liquid System 107

11 Fourier Transform Infrared Spectroscopy as a Tool in Analysis

of Proteus mirabilis Endotoxins 113

12 Laser Interferometry Method as a Novel Tool in Endotoxins Research 125

13 Endotoxin Entrapment on Glass via C-18 Self-Assembled Monolayers

and Rapid Detection Using Drug-Nanoparticle Bioconjugate Probes 133

Prasanta Kalita, Anshuman Dasgupta, and Shalini Gupta

14 A Bioassay for the Determination of Lipopolysaccharides and Lipoproteins 143

Marcus Peters, Petra Bonowitz, and Albrecht Bufe

15 Capillary Electrophoresis Chips for Fingerprinting Endotoxin

Chemotypes and Subclasses 151

Béla Kocsis, Lilla Makszin, Anikó Kilár, Zoltán Péterfi,

and Ferenc Kilár

16 Micromethods for Isolation and Structural Characterization

of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides 167

Alexey Novikov, Aude Breton, and Martine Caroff

17 Mass Spectrometry for Profiling LOS and Lipid A Structures

from Whole-Cell Lysates: Directly from a Few Bacterial Colonies

or from Liquid Broth Cultures 187

Béla Kocsis, Anikó Kilár, Szandra Péter, Ágnes Dörnyei, Viktor Sándor,

and Ferenc Kilár

Index 199

elisabeth carNiel • Institut Pasteur, Unité de Recherche Yersinia, Paris, France

MartiNe caroFF • LPS-BioSciences, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Orsay, France

University, Taichung, Taiwan

JérôMe cheNau • CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Yvette, France

Gif-sur-Patrick cherubiN • Burnett School of Biomedical Sciences, College of Medicine, University

of Central Florida, Orlando, FL, USA

GrzeGorz czerwoNka • Department of Microbiology, Jan Kochanowski University, Kielce, Poland

Jessica daNiel • Department of Chemistry, Metropolitan State University of Denver, Denver, CO, USA

aNshuMaN dasGuPta • Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, RWTH Aachen University Clinic, Aachen, Germany

Mahesh dharNe • NCIM Resource Centre, CSIR-National Chemical Laboratory (NCL), Pune, Maharashtra, India

ÁGNes dörNyei • Department of Analytical and Environmental Chemistry, University of Pécs, Pécs, Hungary

saleM elkahoui • Laboratoire des Substances Bioactives, Le Centre de Biotechnologie à la Technopole de Borj-Cédria, Hammam-Lif, Tunisia

FraNçois FeNaille • CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Gif-sur-Yvette, France

Contributors

lisa Fetter • Department of Chemistry, Metropolitan State University of Denver, Denver,

CO, USA

soFia Filali • Institut Pasteur, Unité de Recherche Yersinia, Paris, France

Markus Fischer • Hamburg School of Food Science, Institute of Food Chemistry, University

of Hamburg, Hamburg, Germany

christiN Fischer • Hamburg School of Food Science, Institute of Food Chemistry,

University of Hamburg, Hamburg, Germany

kushal N Gohil • NCIM Resource Centre, CSIR- National Chemical Laboratory (NCL), Pune, Maharashtra, India

shaliNi GuPta • Department of Chemical Engineering, Indian Institute of Technology, Delhi, India

XiaoFei hu • Henan Academy of Agriculture Science/Key Laboratory of Animal

Immunology, Ministry of Agriculture/Henan Key Laboratory of Animal Immunology, Zhengzhou, China

CO, USA

christoPhe JuNot • CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Gif-sur-Yvette, France

wiesław kaca • Department of Microbiology, Jan Kochanowski University, Kielce, Poland

PrasaNta kalita • Department of Chemical Engineering, Indian Institute of Technology, Delhi, India

FereNc kilÁr • Institute of Bioanalysis, Faculty of Medicine and Szentágothai Research Center, University of Pécs, Pécs, Hungary

University, Taipei, Taiwan

University, Taichung, Taiwan

biswaJit Maiti • Division of Food Hygiene, Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

lilla MaksziN • Institute of Bioanalysis, Faculty of Medicine, University of Pécs, Pécs, Hungary

takashi MatsuMoto • R&D Center, NH Foods Ltd , Ibaraki, Japan

FuMiki MoriMatsu • Center for Regional Collaboration in Research and Education, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

aleXaNdra Morlay • University Grenoble Alpes, SyMMES UMR 5819, CNRS, SyMMES UMR 5819, CEA, SyMMES UMR 5819, Grenoble, France

basavraJ NaGoba • Maharashtra Institute of Medical Sciences & Research (Medical College), Latur, Maharashtra, India

beatriz QuiñoNes • USDA-ARS, Produce Safety and Microbiology Research Unit,

Western Regional Research Center, Albany, CA, USA

yoaNN rouPioz • University Grenoble Alpes, SyMMES UMR 5819, CNRS, SyMMES UMR 5819, CEA, SyMMES UMR 5819, Grenoble, France

5819, CEA, SyMMES UMR 5819, Grenoble, France

teisha J rowlaNd • Cardiovascular Institute and Adult Medical Genetics Program, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, USA

ryuta sakaMoto • R&D Center, NH Foods Ltd , Ibaraki, Japan

viktor sÁNdor • Faculty of Medicine, Szentágothai Research Center, Institute of

Bioanalysis, University of Pécs, Pécs, Hungary

stéPhaNie siMoN • CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse,

Gif-sur-Yvette, France

bożeNa szerMer-olearNik • Laboratory of Biomedical Chemistry - "Neolek," Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of

Sciences, Wroclaw, Poland

viNceNt teMPlier • University Grenoble Alpes, SyMMES UMR 5819, CNRS, SyMMES UMR 5819, CEA, SyMMES UMR 5819, Grenoble, France

Central Florida, Orlando, FL, USA

hervé vollaNd • CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Yvette, France

Gif-sur-sławoMir wąsik • Department of Molecular Physics, Jan Kochanowski University, Kielce, Poland

eiki yaMasaki • Division of Food Hygiene, Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

wallace yokoyaMa • USDA-ARS, Healthy Processed Foods Research Unit, Western Regional Research Center, Albany, CA, USA

PauliNa żarNowiec • Department of Microbiology, Jan Kochanowski University, Kielce, Poland

GaiPiNG zhaNG • Henan Academy of Agricultural Science/Key Laboratory of Animal Immunology, Ministry of Agriculture/Henan Key Laboratory of Animal Immunology, Zhengzhou, China

Contributors

Otto Holst (ed.), Microbial Toxins: Methods and Protocols, Methods in Molecular Biology, vol 1600,

DOI 10.1007/978-1-4939-6958-6_1, © Springer Science+Business Media LLC 2017

As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139

infection, detection of CT is an important biomarker for diagnosis of the disease The procedure for

patho-genicity analysis of V cholerae isolates must be carefully developed for the reason that the amount of CT produced by V cholerae varies according to the medium used and culture conditions (i.e temperature and

aeration status) applied Here we describe a reproducible rapid method for analysis of CT production by

toxigenic V cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of

purified recombinant CT.

Key words Immunochromatographic test strip, V cholerae, Cholera toxin, Toxigenicity, Rapid

diag-nostic tests, AKI medium

1 Introduction

Cholera remains a major public health problem, especially in oping countries, and the seventh pandemic of cholera, which began

devel-in 1961 is still ongodevel-ing In the case of diagnosis of cholera, after or

along with the detection of the causative agent Vibrio cholerae,

veri-fication of cholera toxin (CT) production is of added significance,

because only V cholerae produces CT which is responsible for

chol-era symptoms such as acute “rice watery” diarrhea Various ods, including immunoassays like immunochromatography (IC), enzyme-linked immunosorbent assay (ELISA), reversed passive latex agglutination (RPLA) etc., DNA-based assays like polymerase chain reaction (PCR), quantitative PCR (qPCR), loop mediated isothermal amplification (LAMP) etc., and bioassays including rab-bit ileal loop test, rabbit skin test, cultured Chinese hamster ovary

meth-(CHO) cell assay, etc for toxigenicity investigation of V cholerae

DNA-based assays contribute to the rapid detection of CT and facilitate

timely, in some cases, on-site responses While DNA-based assays may be more sensitive than immunoassays, the latter have an impor-tant advantage for the detection of extracellular bacterial toxin and analysis of the toxin expression level Recently, some novel method-ology of immunoassays with extremely high sensitivity has been

utilized immunoassay because it is rapid and very easy to conduct When IC is used, careful consideration has to be given to the way samples are prepared to allow an optimal production of the target protein

Previously, we have established a toxigenic V cholerae-specific

detect CT in V cholerae cultures in which at least 10 ng/mL of CT was expressed The amount of CT produced in V cholerae El Tor,

pan-demic varies according to the medium used and culture conditions (i.e temperature and aeration status), and the optimal condition is significantly different from that for the classical biotype which was

medium is one of the most efficient media to induce CT expression

in V cholerae El Tor It was reported that if V cholerae El Tor

strains were cultured in AKI medium under biphasic culture tions, i.e 4 h cultivation in a stationary phase followed by 16 h cultivation in a shaking flask at 37 °C, most of the strains produced

CT-IC with AKI medium is advantageous in analyzing the ability

of V cholerae isolates to produce CT.

2 Materials

Prepare all media and solutions using ultrapure water (deionized)

at room temperature

cylin-der Add distilled water to a volume of 20 mL Dissolve all

addi-tional distilled water Filter the solution by using DISMIC Mixed Cellulose Ester Syringe Filter Unit (25AS Type) having

2 AKI medium: Weigh 4.5 g of Bacto™ Peptone, 1.2 g of yeast extract and 1.5 g of NaCl and transfer to 300 mL glass beaker containing about 200 mL of distilled water Dissolve all pow-der completely Transfer the solution to 300 mL graduated cylinder and make up to 282 mL with distilled water Transfer the solution into the autoclavable container and sterilize the

2.1 Culture Media

Eiki Yamasaki et al.

solution with autoclave unit at 121 °C for 15 min After ing the sterilized medium to room temperature, add 18 mL of

3 Luria-Bertani (LB) agar plate: Weigh 4.0 g of Bacto™ Tryptone, 2.0 g of Bacto™ Yeast extract and 4.0 g of NaCl and transfer to 500 mL beaker containing about 350 mL of dis-tilled water Dissolve all powder completely Transfer the solu-tion to 500 mL graduated cylinder and make up to 400 mL with distilled water Transfer the solution into the autoclavable container containing 6.0 g of agar (powder) After mixing the solution, sterilize with autoclave unit at 121 °C for 15 min After cooling the sterilized medium to 50–55 °C, pour it into sterile Petri plates

1 The test strip was prepared with rabbit polyclonal antibodies raised against recombinant purified CT as reported previously

3 Methods

1 Inoculate a V cholerae isolate (isolated by the established

pro-cedures) onto a non-selective agar medium such as LB agar

2 Incubate the LB agar plate at 37 °C for 18–24 h until colonies can be observed

3 Pick a well isolated colony and inoculate a culture tube

4 Let the tube stand at 37 °C for 4 h

5 Transfer entire culture into a sterilized 100 mL Erlenmeyer

6 Incubate in the shaking incubator at 37 °C for about 16 h

7 The obtained bacterial cell culture is used in the analysis with

3 Leave the strip for about 15 min at room temperature

4 Observe the test result A positive result shows reddish purple lines in the test position and control position A negative result shows a reddish purple line only in the control position If no reddish purple line appears in the control position, the result is considered to be invalid

2 Put the CT-IC in the micro tube to immerse the sample

3 Leave the strip for about 15 min at room temperature

4 Observe the test result Criteria for result judgment are same as

3 Selective or differential media such as TCBS agar, Vibrio agar

or CHROMagar™ Vibrio can be used for isolation of V erae Before the analysis with CT-IC, it is better to do subcul-

chol-tivation on non-selective agar medium to obtain completely isolated colony

Fig 1 Illustration of an immunochromatographic test strip for detection of CT

Sample application section b, Reagent containing section c, Detection section

d, Absorbent pad e, Test line for CT detection position f, Control line appearance

position

Eiki Yamasaki et al.

4 For the stationary cultivation phase, a small sterilized container such as 15 mL centrifuge tube (height, 150 mm; diameter,

15 mm) can be used Degassing is not needed

5 For the shaking cultivation phase, Erlenmeyer flask with the volume more than 100 mL (i.e more than 10 times of volume

of the culture) must be used to enforce adequate aeration

6 Expression of CT not only in V cholerae O1 El Tor but also in

other serotypes is known to vary depending on the culture conditions AKI medium is known as an effective medium to induce CT expression Two culture conditions are known for effective induction of CT expression in AKI medium: AKI-SW condition (4 h cultivation in a stationary test tube followed by

>16 h cultivation in a shaking flask at 37 °C) and AKI

V cholerae strains revealed that CT expression under AKI-SW

condition was considerably higher than under AKI condition

Fig 2 Expression of CT under AKI-SW and AKI conditions Concentration of CT in the culture supernatant were

obtained after cultivation of various V cholerae isolates under AKI-SW (black bar) or AKI (gray bar) and were

experiments

the analysis with CT-IC Among the 15 isolates we analyzed,

concen-tration of substantially lower than the detection limit of CT-IC (10 ng/mL) under AKI condition However, CT expression could be detected by CT-IC with AKI-SW condition (concen-tration of CT was 0.28 ± 0.12 ng/mL in AKI condition whereas 16.1 ± 6.97 ng/mL in AKI-SW condition)

7 We confirmed that centrifugation to remove bacterial cells did

(moderate CT expression with low cell density), case No 2 (high CT expression with high cell density) and case No 3 (low CT expression with high cell density), whole cell cultures and cleared supernatants that were obtained after centrifuga-

tion (900 × g, 5 min) gave the same results in CT-IC analysis

These results indicated that bacterial cells did not inhibit tions developing on the immunochromatographic test strip In

reac-addition, in case No 4, ct gene-negative V cholerae El Tor

Ogawa strain with high cell density did not give false-positive results even if the whole cell culture was applied to the immu-nochromatographic test strip

8 The test strips are normally provided with light shielding age and stored at 4 °C Allow the test strips to come to room temperature (20–25 °C) before opening the package to pre-vent moisture absorption Do not touch with bare fingers on

pack-Table 1 Effect of centrifugation before immunochromatographic analysis on CT-IC results

*1: The strain No are matched with the number in Fig 2

*2: The concentration of CT in the clear supernatant of the cultures measured with bead-ELISA are cated Data are mean ± SD of values from three independent experiments

indi-*3: Relative cell density and pictures of cell cultures in cuvettes are shown

*4: Results of CT-IC analyses are shown The “+++”, “+” or “-” symbols are placed on the left side of the strips developing “strong”, “faint” or “no” bands at test lines respectively T: test line, C: control line Eiki Yamasaki et al.

the sample application section and detection section (Fig 1)

It is better to hold the absorbent pad with tweezers or gloved fingers when handling the test strips

9 Be careful not to overload the sample application zone to vent spillage Apply the culture in two batches as appropriate

sample below the reagent containing sections of the test strips

Acknowledgements

This study was supported in part by a Grant- in- Aid of Ministry of Health, Labor and Welfare (H26-Shinkou-Shitei-002)

References

1 Dick MH, Guillerm M, Moussy F et al (2012)

Review of two decades of cholera diagnostics—

how far have we really come? PLoS Negl Trop

Dis 6:e1845

2 CDC (1999) Laboratory methods for the

diag-nosis of Vibrio cholerae Chapter VII

3 Palchetti I, Mascini M (2008) Electroanalytical

biosensors and their potential for food

patho-gen and toxin detection Anal Bioanal Chem

391:455–471

4 Shlyapnikov YM, Shlyapnikova EA, Simonova

MA et al (2012) Rapid simultaneous

ultrasen-sitive immunodetection of five bacterial toxins

Anal Chem 84:5596–5603

5 Yamasaki E, Sakamoto R, Matsumoto T et al

(2013) Development of an

immunochromato-graphic test strip for detection of cholera toxin

Biomed Res Int 2013:679038

6 Iwanaga M, Kuyyakanond T (1987) Large

production of cholera toxin by Vibrio cholerae

O1 in yeast extract peptone water J Clin

Microbiol 25:2314–2316

7 Iwanaga M, Yamamoto K (1985) New medium

for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor J Clin Microbiol

22:405–408

8 Iwanaga M, Yamamoto K, Higa N et al (1986) Culture conditions production for

stimulating cholera toxin by Vibrio cholerae

OI El Tor Microbiol Immunol 30:1075– 1083

9 Sánchez J, Medina G, Buhse T et al (2004) Expression of cholera toxin under non-AKI

conditions in Vibrio cholerae El Tor induced by

increasing the exposed surface of cultures

J Bacteriol 186:1355–1361

10 Yonekita T, Fujimura T, Morishita N et al (2013) Simple, rapid, and reliable detection of

Escherichia coli O26 using

immunochromatog-raphy J Food Prot 76:748–754

11 Uesaka Y, Otsuka Y, Kashida M et al (1992) Detection of cholera toxin by a highly sensitive linked immunosorbent assay Microbiol Immunol 36:43–53

Otto Holst (ed.), Microbial Toxins: Methods and Protocols, Methods in Molecular Biology, vol 1600,

DOI 10.1007/978-1-4939-6958-6_2, © Springer Science+Business Media LLC 2017

Chapter 2

Electrochemical Aptamer Scaffold Biosensors

for Detection of Botulism and Ricin Proteins

Jessica Daniel, Lisa Fetter, Susan Jett, Teisha J Rowland,

and Andrew J Bonham

Abstract

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of lar targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

molecu-Key words Biosensors, Toxins, Electrochemical, Aptamer, Botulism, Ricin, Voltammetry, E-DNA,

Gold electrodes, Proteins

1 Introduction

Accurate and rapid detection of biomarkers is useful in many

Biosensors, which are devices that incorporate biological

suited to overcoming challenges associated with detecting a

(e.g., whole blood or river water samples) In addition, biosensors have several other appealing features that allow them to be used successfully in unique and challenging situations, including high specificity of detection, high reproducibility, relative ease of manu-facturing and affordability, rapid throughput, direct readout, and minimal invasiveness

One prominent and successful class of biosensors is

rely on the changing conformational dynamics of a synthetic

deoxyoligonucleotide (DNA) scaffold containing an aptamer or transcription factor-binding motif that recognizes the target

functional groups to enable attachment to an electrode surface (typically through a thiol-gold bond) and to an electrochemi-

When the biosensor is subjected to voltammetric analysis, the scaffold conformation changes depending on whether or not it

is bound to its target biomolecule, and this affects the dynamics and the position (and thus observed current) of the electro-chemically active reporter molecule relative to the electrode

In theory, E-DNA biosensors can be designed to detect any molecule for which oligonucleotide-binding interactions are known or discoverable (such as via systematic evolution of ligands

by exponential enrichment [SELEX]) Recently, our group ated E-DNA biosensors for the detection of protein toxins respon-

design and use of novel E-DNA biosensors against these and lar targets The biosensors described here can detect nanomolar concentrations of ricin chain A and botulinum neurotoxin variant

and function when challenged with complex matrices such as blood

Fig 1 Schematic of E-DNA biosensor, illustrating the change in position and dynamics of the reporter molecule

(methylene blue, represented by a blue star) attached to the DNA scaffold in response to binding of the

mono-layer of 6-mercapto-1-hexanol (not shown) to prevent nonspecific binding of biomolecules (Reproduced from

2 Materials

Prepare all solutions using ultrapure water (prepared by purifying

analytical grade reagents Prepare and store all reagents at room temperature (unless indicated otherwise) Diligently follow all waste disposal regulations when disposing of waste materials

Fig 2 Representative dose-responsive curves of peak current vs toxin concentration for botulinum neurotoxin

Fig 3 The botulinum (BoNTA) and ricin (RTA) biosensors display minimal off-

target responses when challenged with off-target proteins, including bovine serum albumin (BSA) and other biomolecular targets, such as the unrelated DNA-

binding protein complex Myc/Max Student’s t-test was performed to compare on-target to off-target response (* for p < 0.05, *** for p < 0.0001) (Reproduced

Protein Toxin E-DNA Biosensors

1 Biosensor DNA: Synthetic DNA scaffold with 5′ terminal

and internal thymidine-methylene blue to be used as an electrochemically active reporter molecule (methylene blue

wrapped in aluminum foil The ricin biosensor sequence used

GGG AA CGG AGT GGT CCG TTATTA ACC ACT ATTT

ATTTGACACT TT TCAAAC T GTCCTATGAC A GTCCA

2 Quickfold application from the DINAMelt web server, hosted

by the RNA Institute at the State University of New York at

DINAMelt/Quickfold (see Note 2).

http://www.bonhamlab.com/wp-content/uploads/2016/05/Fealden-0.2_04232016.zip (see Note 3).

4 PCR tubes: 0.5 mL flat-cap PCR tubes, RNase- and DNase- free, polypropylene

1 Pine Research Instrumentation WaveNano USB Potentiostat

4 Pine Research Instrumentation Compact Voltammetry Cable

5 Pine Research Instrumentation Ceramic Patterned Gold Electrode

6 Pine Research Instrumentation AfterMath Scientific Data Organizer Software

7 Alkaline cleaning solution: 0.5 M NaOH

1 TCEP solution: 1 M Tris(2-carboxyethyl)phosphine

2 Phosphate-buffered saline (PBS): 8 g NaCl, 0.2 g KCl, 1.44 g

adjusted to 1 L with ultrapure water

3 Mercaptohexanol solution: 0.001 M 6-mercapto-1-hexanol in PBS Prepare and work with solution in a chemical fume hood Store at 4 °C for up to 1 month

4 PCR tubes: 0.5 mL flat-cap PCR tubes, RNase- and DNase- free, polypropylene

5 Petri dish: 100 mm × 15 mm, polystyrene

2 Ricin solution: Ricin A chain from Ricinus communis

(castor-bean or castor-oil-plant, from Sigma-Aldrich) Resuspend at

1 mg/mL in PBS Store aliquoted at 4 °C

3 Botulism solution: Botulinum neurotoxin variant A1 atoxic

4 AnyPeakFinder software program (source code available at

http://www.bonhamlab.com/tools/code/) or AfterMath

2 Identify regions of the motif that are presumed to be

detailed mechanistic binding studies are often available in the literature; the regions of interest will typically be predicted to form “loops” in their secondary structure Confirmation via Quickfold may be useful

3 Design a synthetic DNA scaffold that incorporates the motif region(s) identified to be essential for target binding interactions and allows for potential disruption of these bind-ing interactions To do this, design the essential regions to be

deoxyoli-gonucleotides that are partially complementary to the tial regions, facilitating the formation of secondary structures

or folding patterns that likely disrupt target binding Multiple rounds of confirmation via Quickfold or other secondary

4 Continue designing the scaffold by iteratively adding, ing, or changing oligonucleotides in the nonessential regions

remov-to ultimately create a scaffold with two potential, equally able (i.e., isoenergetic) states: one state in which the essential regions are available for target binding interactions (i.e., in their native form) and one in which the essential regions are unavailable due to being base paired with nonessential regions

design-ing tool that may be used to help automate this process

5 Once a scaffold has been designed with the two desirable isoenergetic states, modify the scaffold design to include an

terminus of the entire scaffold should serve as the attachment point by forming a thiol-gold bond between the scaffold and the gold electrode surface

6 Further modify the scaffold design to include an cally active reporter molecule; here a methylene blue is used The methylene blue can be easily covalently appended to a modified thymine Examine the scaffold’s two isoenergetic states to identify a thymine that is nonessential in a significantly different folded environment and has significant distance

electrochemi-Fig 4 Schematic of biosensor design workflow process An initial aptamer is truncated to essential regions

and then flanked by a random scaffold of novel oligonucleotides Secondary structure predictions are used to guide changes to the scaffold sequence to promote the formation of isoenergetic states that either present or obscure the aptamer essential regions The addition of a reporter molecule (e.g., methylene blue, “MB”) and surface attachment modifications (i.e., thiol-gold bond, “HS”) leads to a completed biosensor design

7 Synthesize the designed scaffold using a DNA synthesis pany or in-house phosphoramidite deoxyoligonucleotide synthesis

8 Resuspend the DNA in ultrapure water upon receipt at a

1 Connect the WaveNano USB Potentiostat to a computer via a USB cable

2 Connect the Compact Voltammetry Cell Grip Mount to the potentiostat using the WaveNano Shielded Cell Cable and Compact Voltammetry Cable, being sure that the alligator clips of the Shielded Cell Cable do not touch each other

3 Place the Ceramic Patterned Gold Electrode face up in the grip mount, and add a plastic adaptor spacer (included with elec-trode) at the bottom of the grip mount to ensure solid contact between the grip mount and electrode Ensure that the black ground electrode of the Shielded Cell Cable is connected to

4 Power on the potentiostat and ensure that the status light is green

5 Open and log in to the AfterMath Scientific Data Organizer Software Ensure that the WaveNano Potentiostat is recognized

3.2 Electrode

Preparation

Fig 5 Image of Pine Research Instrumentation (a) WaveNano instrument with

surfaces The biosensor attaches to the central, circular gold electrode

Protein Toxin E-DNA Biosensors

and communicating with AfterMath; the potentiostat’s status should be listed as “idle” (see AfterMath support site for guid-

6 Insert the electrode into a 30 mL beaker, and add 15 mL of alkaline cleaning solution, ensuring that the exposed gold sur-faces of the electrode are submerged and the grip mount and contacts on the electrode remain dry

7 Create and run a new cyclic voltammetry experiment to

2 V/s This will reductively desorb any sulfur-linked molecules

on the electrode surface

8 Remove the electrode from the alkaline cleaning solution,

acid cleaning solution (instead of alkaline cleaning solution)

9 Create and run a new bulk electrolysis experiment to perform oxidation using 2 V applied for 5 s followed by reduction

contaminants and then reduce any gold oxide formed

10 Create and run a new cyclic voltammetry experiment to form cyclic oxidation and reduction voltammetric scans, per-forming 20 scans with a scan rate of 4 V/s, followed by a further 4 scans at 0.1 V/s, from 0.35 V to 1.5 V This step will sequentially oxidize and then reduce any remaining contami-nants on the electrode surface

11 Remove the electrode from the acid cleaning solution, rinse

solution (instead of alkaline cleaning solution)

12 Create a new cyclic voltammetry experiment, and perform scans over four different potential ranges, each for ten scans at scan rate of 0.1 V/s: 0.2–0.75 V, 0.2–1.0 V, 0.2–1.25 V, and 0.2–1.5 V This will etch away the surface layer of the electrode

as gold chloride complexes, resulting in a substantially cleaned surface

13 Remove the electrode from the etch solution, rinse with

solu-tion (instead of alkaline cleaning solusolu-tion)

14 Create a new cyclic voltammetry experiment, and perform

This will oxidize a gold oxide layer on the electrode and then completely reduce it The area under the reduction peak can be used to calculate the available surface area of

15 Store the cleaned electrode submerged in evaluation solution for

Avoid exposing the sensor DNA to light

solution Allow the sensor DNA/TCEP mixture to react for at least 15 min, until it has changed from light blue to clear in

15 of Subheading 3.3) and rinse it with ultrapure water Using

a clean, delicate task wiper (e.g., a Kimwipe), dry the electrode

by wicking it dry, touching only the ceramic portions of the electrode and taking care not to touch the exposed gold surfaces

the electrode’s surface, being careful to cover the entire exposed gold surface Place the electrode inside a closed petri dish for

60 min, which will minimize evaporation and allow the reaction

to proceed In arid climates, we have found it is important to

the electrode on top of a small, upside-down weigh boat in the

6 Using a delicate task wiper, dry the electrode as described in

step 4 Immediately proceed with the next step to prevent the

electrode from completely drying

elec-trode, being careful to cover the entire exposed gold surface Place the electrode inside a petri dish Allow the reaction to

8 Equilibrate the prepared biosensor in PBS for at least 20 min

(instead of alkaline cleaning solution)

1 Remove aliquoted protein solution (either ricin or botulinum solution, depending on desired target biomolecule) from stor-age and place on ice

2 In microcentrifuge tubes, prepare a total of approximately ten serial dilutions of the protein solution in PBS, each dilution

dilutions on ice and use them within 2 h

remove the electrode from the PBS Using a delicate task wiper,

2 Place the grip mount and electrode in a horizontal position with the exposed gold surfaces facing up.

PBS, being careful to cover the working, counter, and ence elements of the electrode Allow the electrode to

refer-equilibrate for at least 10 min Note: Instead of PBS, bovine

blood serum or whole bovine blood may alternatively be

4 Ensure that the Compact Voltammetry Cable and Shielded Cell Cable are correctly attached to the grip mount and that the potentiostat’s status in the AfterMath software is shown as

“idle.”

5 Create a new square wave voltammetry experiment in the

an amplitude of 50 mV, and a step size of 1 mV The optimal square wave frequency should be experimentally derived as it

100 Hz is a typically useful frequency for a wide variety of biosensors

6 Run the experiment; a rounded peak in current at

is proportional to the effective efficiency of electron transfer

7 Rinse electrode with ultrapure water Using a delicate task wiper,

0.01 nM ricin or botulinum solution (prepared in Subheading

Fig 6 Square wave voltammograms of BoNTA biosensor equilibrated in PBS with

permission of the Royal Society of Chemistry)

elements of the electrode Allow the electrode to equilibrate for at least 10 min

to 0.1 V with an amplitude of 50 mV and a step size of 1 mV using the experimentally determined optimal frequency

10 Run the experiment; the methylene blue-derived peak in

mag-nitude of any change in peak height reflects changes in biosensor signaling due to the presence of the target biomolecule

increas-ing concentration For each dilution, measure the peak in rent using AfterMath’s peak height tool, or export the data as

cur-a commcur-a-sepcur-arcur-ated vcur-alue (csv) file formcur-at, cur-and use the AnyPeakFinder program to determine the peak heights

cur-rent, calculate the relative change in current for each dilution

as a percentage increase or decrease in signal For example, for each dilution, the baseline peak height could be subtracted from the dilution’s peak height, and this value could be divided

by the baseline peak height to calculate the percentage change

13 Use the resulting data to construct a saturation binding curve, allowing visualization of the apparent dissociation constant for the target

14 Following establishment of the target concentration dent response, this section’s procedure can be repeated using samples of unknown protein concentration to allow for quan-tification of the protein concentration in solution, enabling biosensing applications

depen-4 Notes

1 DNA synthesis is performed by standard phosphoramidite pling on a solid support, which is available from many compa-nies, such as Biosearch Technologies or Integrated DNA

deoxynucleotide phosphoramidite is attached to a controlled

then used to remove DMT, followed by coupling to the next deoxynucleotide phosphoramidite, protective acetylation, and oxidation and then a repeated cycle of deprotection and cou-pling Modified deoxynucleotide phosphoramidites can be easily included in this synthesis process

Protein Toxin E-DNA Biosensors

2 The online Quickfold module is convenient, but there are eral other tools available that predict DNA secondary structure folding, and any of these other tools should, in principle, be sufficient for the necessary analysis Examples include RNAstructure from the University of Rochester Medical

Servers/Predict1/Predict1.html) and Integrated DNA

idtdna.com/calc/analyzer)

3 The Fealden software significantly automates the task of ating predicted DNA secondary structures for correct biosen-sor conformational states, but it is optional and may require Python programming experience to customize it for new appli-cations Fealden requires a UNIX-like environment and has been confirmed to work on Ubuntu Linux and Mac OSX

4 Potentiostats and analysis software are available from several vendors; here we use Pine Research Instrumentation Other vendors that could provide suitable instrumentation packages include CH Instruments, Inc., and Metrohm Autolab Nova

5 Analysis of square wave voltammetric data requires accurately measuring the height of observed current peaks (when plot-ting current vs voltage) AfterMath software includes a man-ual tool for this measurement, and as the data can be exported

in csv format, a variety of computational tools can be used to identify and measure current peaks, including Mathematica and Matlab Our lab provides source code for AnyPeakFinder,

a Python program that can automatically read csv formats and

tools/code/any-peak-finder-interactive/

6 The core principles of selecting correct regions and optimizing folded structures have been explored in a number of studies

approach can often yield good results Generally, structures with predicted free energies within 1 kJ/mol are more likely to

be meaningful Minimizing the number of predicted states helps avoid inconsistent results

7 For sensor DNA solutions, the DNA sensor concentration is

TCEP solution must be sufficient to fully reduce the disulfide modification present in the sensor DNA solution, and conse-quently in this protocol the amount of TCEP solution added is

in high excess The observed color change is due to reversible reduction of the methylene blue modification Although this change has no impact on the final performance of the biosen-sor, it is a convenient marker for the progress of reduction of the solution

8 This process allows the sensor DNA to attach to the surface in

an incomplete monolayer, with average spacing between ecules that minimizes or eliminates interactions between neighboring sensors, which is important for reproducible per-formance Optimizations of this surface packing have been

9 The mercaptohexanol solution addition acts to form a stable, mixed surface monolayer with the attached sensor DNA While 6-mercapto-1-hexanol is the most common of these “passiv-ation” chemicals, our lab has additionally found success with the use of (11-mercaptoundecyl)tetra(ethylene glycol), which presents a more biocompatible monolayer for studies in com-plex matrices The monolayer formed prevents nonspecific interactions of the biomolecule target with the electrode’s gold surface and provides a more reproducible current response

fol-lowed by sealing the electrode in a petri dish and storing it overnight at 4 °C, has also been successful Our lab has also

electrodes with saline sodium citrate (SSC) buffer or 1% bovine serum albumin (BSA) buffer with minimal successes This was performed by allowing the mercaptohexanol to adhere to the electrode overnight, then removing it with a pipette, and add-

to sit for 10 min before beginning trials

10 To serve as a test bed for complex matrices uses of these sors, we have employed both adult bovine serum and bovine whole blood (citrate stabilized) in place of PBS In both matri-ces, sensors still performed well, although the magnitude of current changes is often reduced

11 The precise voltage where the peak in current is found for methylene blue will vary based on solution conditions (e.g.,

pH and ionic content) Different reporter dyes will have a ferent characteristic voltage for peak current

pro-Protein Toxin E-DNA Biosensors

1 Miranda-Castro R, de-los-Santos-Álvarez N,

Lobo-Castañón MJ (2016) Aptamers as synthetic

receptors for food quality and safety control

Compr Anal Chem 74:155–191 doi: 10.1016/

bs.coac.2016.03.021

2 Ferguson BS, Hoggarth DA, Maliniak D et al

(2013) Real-time, aptamer-based tracking of

circulating therapeutic agents in living animals

Sci Transl Med 5:213ra165

3 Lee TM-H (2008) Over-the-counter biosensors:

past, present, and future Sensors 8:5535–5559

4 Lubin AA, Lai RY, Baker BR et al (2006)

Sequence-specific, electronic detection of

oli-gonucleotides in blood, soil, and foodstuffs

with the reagentless, reusable E-DNA sensor

Anal Chem 78:5671–5677

5 Hasanzadeh M, Shadjou N (2016)

Electrochemical nanobiosensing in whole

blood: recent advances TrAC Trends Anal

Chem 80:167–176

6 Lubin AA, Plaxco KW (2010) Folding-based

electrochemical biosensors: the case for

respon-sive nucleic acid architectures Acc Chem Res

43:496–505

7 Vallee-Belisle A, Bonham AJ, Reich NO et al

(2011) Transcription factor beacons for the

quantitative detection of DNA binding activity

J Am Chem Soc 133:13836–13839

8 Schaffner SR, Norquest K, Baravik E et al

(2014) Conformational design optimization of

transcription factor beacon DNA biosensors

Sens Bio-Sensing Res 2:49–54

9 Fetter L, Richards J, Daniel J et al (2015)

Electrochemical aptamer scaffold biosensors

for detection of botulism and ricin toxins

Chem Commun (Camb) 51:15137–15140

10 Rowe AA, White RJ, Bonham AJ, Plaxco KW

(2011) Fabrication of electrochemical-DNA

biosensors for the reagentless detection of

nucleic acids, proteins and small molecules

J Vis Exp 52:e2922

11 Ricci F, Plaxco KW (2008) E-DNA sensors for

convenient, label-free electrochemical detection

of hybridization Microchim Acta 163:149–155

12 Xiao Y, Uzawa T, White RJ et al (2009) On the

signaling of electrochemical aptamer-based

sensors: collision- and folding-based

mecha-nisms Electroanalysis 21:1267–1271

13 Liu J, Wagan S, Dávila-Morris M et al (2014)

Achieving reproducible performance of

elec-trochemical folding aptamer-based sensors on

microelectrodes: challenges and prospects

Anal Chem 86:11417–11424

14 Xiao Y, Rowe AA, Plaxco KW (2007)

Electrochemical detection of parts-per-billion

lead via an electrode-bound DNAzyme bly J Am Chem Soc 129:262–263

15 Vallée-Bélisle A, Ricci F, Uzawa T et al (2012) Bioelectrochemical switches for the quantita- tive detection of antibodies directly in whole blood J Am Chem Soc 134:15197–15200

16 Bonham AJ, Hsieh K, Ferguson BS et al (2012) Quantification of transcription factor binding

in cell extracts using an electrochemical, structure- switching biosensor J Am Chem Soc 134:3346–3348

17 Markham NR, Zuker M (2005) DINAMelt web server for nucleic acid melting prediction Nucleic Acids Res 33:W577–W581

18 Vazquez-Cintron EJ, Vakulenko M, Band PA

et al (2014) Atoxic derivative of botulinum neurotoxin a as a prototype molecular vehicle for targeted delivery to the neuronal cyto- plasm PLoS One 9:e85517

19 Xiao Y, Lai RY, Plaxco KW (2007) Preparation of electrode-immobilized, redox-modified oligonu- cleotides for electrochemical DNA and aptamer- based sensing Nat Protoc 2:2875–2880

20 Creager SE, Olsen KG (1995) Self-assembled monolayers and enzyme electrodes: progress, problems and prospects Anal Chim Acta 307:277–289

21 White RJ, Plaxco KW (2009) Exploiting binding- induced changes in probe flexibility for the optimization of electrochemical biosen- sors Anal Chem 82:73–76

22 Uzawa T, Cheng RR, White RJ et al (2010) A mechanistic study of electron transfer from the distal termini of electrode-bound, single- stranded DNAs J Am Chem Soc 132:16120–16126

23 Vallée-Bélisle A, Ricci F, Plaxco KW (2012) Engineering biosensors with extended, nar- rowed, or arbitrarily edited dynamic range

J Am Chem Soc 134:2876–2879

24 Vallée-Bélisle A, Plaxco KW (2010) Structure- switching biosensors: inspired by Nature Curr Opin Struct Biol 20:518–526

25 White RJ, Plaxco KW (2009) Engineering new aptamer geometries for electrochemical aptamer-based sensors In: Fell NF, Jr, Swaminathan VS (eds) Proc Soc Photo Opt Instrum Eng SPIE, Department of Chemistry and Biochemistry University of California, Santa Barbara, Santa Barbara, CA 93106-9510,

27 White RJ, Phares N, Lubin AA et al (2008)

Optimization of electrochemical aptamer-

based sensors via optimization of probe

pack-ing density and surface chemistry Langmuir

24:10513–10518

28 Huang K-C, White RJ (2013) Random walk

on a leash: a simple single-molecule diffusion model for surface-tethered redox molecules with flexible linkers J Am Chem Soc 135:12808–12817

Protein Toxin E-DNA Biosensors

Otto Holst (ed.), Microbial Toxins: Methods and Protocols, Methods in Molecular Biology, vol 1600,

DOI 10.1007/978-1-4939-6958-6_3, © Springer Science+Business Media LLC 2017

Chapter 3

A Cell-Based Fluorescent Assay to Detect the Activity

of AB Toxins that Inhibit Protein Synthesis

Patrick Cherubin, Beatriz Quiñones, Salem Elkahoui, Wallace Yokoyama, and Ken Teter

Abstract

Many AB toxins elicit a cytotoxic effect involving the inhibition of protein synthesis In this chapter,

we describe a simple cell-based fluorescent assay to detect and quantify the inhibition of protein synthesis The assay can also identify and characterize toxin inhibitors.

Key words AB toxin, Ricin, Shiga toxin, Toxin detection, Toxin inhibitors, Toxicity assay, Vero cells

1 Introduction

AB-type protein toxins are produced by numerous bacterial

A subunit and a cell-binding B subunit The A and B moieties can encompass different regions of a single polypeptide chain or may represent distinct proteins in various stoichiometries (e.g., AB,

Several methods can detect the toxin-induced inhibition of protein synthesis or resulting cell death A common procedure measures the viability of intoxicated cells by dye exclusion,

require several days of toxin exposure and often involves additional processing steps for data collection Furthermore, as discussed later, there is a temporal disconnect between the inhibition of pro-tein synthesis and the loss of cell viability A direct method to quan-tify the toxin-induced inhibition of protein synthesis measures the incorporation of radiolabeled amino acids into newly synthesized

which is laborious, potentially hazardous, and can only date a limited number of samples Quantitative luciferase-based

assays have been described that are similar to the system reported here, but these systems require several preparatory and/or process-

described assay that monitors the production and secretion of tylcholinesterase likewise requires additional processing steps for

As an alternative to existing technologies, we developed a ple and quantitative cell-based assay for the detection of toxins that inhibit protein synthesis A Vero cell line with constitutive expres-

fluorescent protein (d2EGFP) is challenged with toxin for 18–24 h Intoxicated cells degrade d2EGFP and do not replenish the lost protein due to the toxin-induced block of protein synthesis The fluorescent signal from Vero-d2EGFP cells is accordingly lost in proportion to the applied dose of toxin This assay provides repro-

pro-cedure does not require radioisotopes, commercial kits, or additional processing steps A plate reader is required for reading fluorescent samples, but the only major recurring cost is the use of black-walled, clear-bottom 96-well tissue culture microplates As described below, the noninvasive nature of the fluorescent mea-surement allows the Vero-d2EGFP cells to be used for additional purposes Furthermore, the protocol can be adapted to screen for toxin inhibitors

2 Materials

1 Parental Vero cells (ATCC #CCL-81) and a clonal population

of Vero-2dEGFP cells with stable, constitutive expression of

2 Complete Dulbecco’s Modified Eagle Medium (DMEM) for

(GIBCO), supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 1% antibiotic-antimycotic solution, and 1 mg/mL Geneticin (G-418)

3 Intoxication medium: F-12 + GlutaMAX-I nutrient mixture

4 HyClone antibiotic-antimycotic 100× solution: 10,000 U/mL

amphotericin B (GE Healthcare)

5 Trypsin-EDTA 1× solution containing 0.25% trypsin, 0.9 mM EDTA, and phenol red (GIBCO)

6 HyClone phosphate-buffered saline (PBS) 1× solution:

2.1 Cell Culture

Patrick Cherubin et al.

7 Costar® black-walled 96-well polystyrene plates with a clear,

9 100 × 20 mm tissue culture dishes (Techno Plastic Products)

10 Fisherbrand 25 mL sterile disposable divided well pipette basins (Fisher Scientific)

pipette (p300) (Eppendorf)

1 Ricin (Vector Laboratories, Burlingame, CA)

2 Cell-free culture supernatant containing Shiga toxin 1 (Stx1)

3 Methods

We generated a Vero cell line that stably expresses d2EGFP-N1, an EGFP variant that contains a C-terminal PEST sequence for rapid

state fluorescence in the Vero-d2EGFP cell line is easily detected

syn-thesis, toxin-susceptible cells will degrade d2EGFP and will not produce more of the protein Productive intoxication accordingly

ricin reduced the Vero-d2EGFP fluorescent signal in a dose-

2.2 Toxins

Fig 1 Fluorescent output from the Vero-d2EGFP cells 20,000 parental Vero

(gray) or Vero-d2EGFP (black) cells were subjected to cytofluorometry

was much more dramatic than the loss of cell viability after an 18 h

0.03 ng/mL was recorded by the Vero-d2EGFP assay, whereas the

Both fluorescence and viability were measured in the same cell

the loss of fluorescence after 42 h of toxin exposure, with both

the Vero-d2EGFP system, including (1) relatively rapid detection

of toxin activity, (2) high sensitivity, (3) minimal sample handling for data acquisition, and (4) a noninvasive/nonterminal measure-ment that allows the cells to be used for other purposes such as an MTS assay

To examine how quickly the Vero-d2EGFP assay can detect toxin activity, we monitored the time-dependent decay of EGFP

Using a single concentration of ricin (1 ng/mL), we found the EGFP signal begins to decay 4 h after toxin exposure and contin-ues to decrease until 14 h when a minimal signal of 5–8% is achieved With a 2 h half-life for d2EGFP, a signal strength corresponding to 6% of the unintoxicated control value could the-oretically be reached 8 h after exposure to a toxin that inhibits protein synthesis The longer time frame required to reach this point for ricin-treated cells reflects the temporal delay between toxin binding to the cell surface and A chain delivery to its site of

intoxication in a population of cells These cellular events also

Fig 2 Effect of ricin on protein synthesis and cell viability Fluorescence (filled circles) and cell viability via MTS

incubation with serial dilutions of ricin Results were expressed as percentages of the maximal signal obtained from unintoxicated Vero-d2EGFP cells The means ± standard errors of the means of at least four independent experiments with six replicate samples for each condition are shown

Patrick Cherubin et al.

explain why the loss of EGFP fluorescence is slower in toxin-treated cells than in cells treated with cycloheximide, a membrane-perme-

experiment demonstrated the cellular activity of ricin can be detected 4 h after toxin exposure and reaches its maximal effect on protein synthesis 14 h after toxin exposure

Disruptions to the intoxication process will permit the ued synthesis of d2EGFP in toxin-treated cells This principle was used to identify grape seed extract as a potent inhibitor of both

was then confirmed with an independent toxicity assay that tored the overall level of protein synthesis in cells exposed to Stx2

d2EGFP cells were also protected from ricin, diphtheria toxin, and

Use of the Vero-d2EGFP assay to screen for toxin inhibitors is

com-pounds from grape extract failed to identify any individual inhibitor

extract itself for antitoxin effects Seven polyphenolic fractions, generated by using a modified normal-phase high- performance

cell-free culture supernatant containing Stx1/Stx2 and applied to

Fig 3 Time frame for loss of Vero-d2EGFP fluorescence Measurements of

fluo-rescent intensity were taken at 2 h intervals after incubation of Vero-d2EGFP

cycloheximide (squares) Results were expressed as percentages of the maximal

EGFP signal obtained from untreated Vero-d2EGFP cells The averages ± dard deviations of three independent experiments with six replicate samples per condition are shown

from cells exposed to toxin alone was reduced to 25% of the signal

fluorescence was recorded for Vero-d2EGFP cells challenged with

Fig 4 Use of the Vero-d2EGFP assay to screen for toxin inhibitors (a) A modified method using normal-phase

high-performance liquid chromatography separated grape seed extracts into seven fractions enriched in

were incubated with one of the extract fractions (5% final volume) and a 1:250 dilution of a cell-free culture

supernatant from an E coli strain that expresses both Stx1 and Stx2 After an 18 h incubation, EGFP

fluores-cence was measured with a plate reader Results were expressed as percentages of the maximal EGFP signal obtained from a parallel set of unintoxicated Vero-d2EGFP cells The means ± standard errors of the means of

of epigallocatechin gallate (EgCg) After an 18 h incubation, EGFP fluorescence was measured with a plate reader Results were expressed as percentages of the maximal EGFP signal obtained from a parallel set of unintoxicated Vero-d2EGFP cells The averages ± ranges of two to four independent experiments with six replicate samples per condition are shown For panels B and C, fraction 1 (F1) represents material eluted from

0 to 5 min; F2 represents material eluted from 5 to 10 min; etc

Patrick Cherubin et al.

toxin in the presence of fractions 1–6 However, intoxicated cells co-incubated with fraction 7 retained a stronger fluorescent signal, representing 42% of the unintoxicated control value and a statisti-cally significant difference from the intoxicated control cells

(p = 0.0217, Student’s t-test) A similar screen indicated fractions

screen, a known polyphenol inhibitor of ricin—epigallocatechin

separation of the compounds in fractions 5–7, combined with tional Vero-d2EGFP assays, could potentially identify the specific polyphenols that confer resistance to Stxs and/or ricin

1 Maintain the parental Vero and Vero-d2EGFP cells in a

2 Working in a tissue culture hood, remove the spent medium from the tissue culture dish and wash the cells with 10 mL of sterile 1× PBS

3 Detach cells from the dish by adding 1 mL of trypsin/EDTA

4 Re-suspend detached cells in 9 mL of complete DMEM medium for a total volume of 10 mL

5 Determine the cell concentration with a hemocytometer and alter it to a final concentration of 100,000 cells per mL For the parental Vero cells, the final cell suspension should be in a 1.5 mL volume per 96-well plate For the Vero-d2EGFP cells, the final cell suspension should be in a 9 mL volume per 96-well plate

6 Pour each cell dilution (parental Vero and Vero-d2EGFP) into

a separate sterile basin Using a multichannel pipette (p300),

row (12 wells) of a black-walled 96-well microplate with clear bottom Again using a p300 multichannel pipette, transfer

remaining wells of the microplate

1 Prepare several ten- or twofold serial dilutions of toxin in serum- free Ham’s F-12 medium The final volume for each toxin dilution is 0.8 mL for 6 replicate samples or 1.5 mL for

12 replicate samples For a screen of toxin inhibitors, prepare identical serial dilutions in Ham’s F-12 medium containing the final concentration of inhibitor

2 Remove medium from the Vero and Vero-d2EGFP cells with

a multi-well vacuum aspirator

free Ham’s F-12 medium, which is added with a p300 channel pipette

serial dilutions in the absence or presence of inhibitor

5 As an unintoxicated control condition, incubate Vero-d2EGFP

F-12 medium When inhibitor screens are performed, bate additional sets of unintoxicated Vero-d2EGFP cells with

6 As a positive control for the loss of fluorescence, treat one set

of unintoxicated Vero-d2EGFP cells with the protein synthesis inhibitor cycloheximide

humidified incubator before fluorescence measurements are taken

of 1× PBS

PBS to the cells Read measurements from cells bathed in 1×

4 Measure the EGFP fluorescence on a Synergy H1 Multi-Mode Microplate Reader (BioTek, Winooski, VT) with bottom optics position using a 485 nm excitation and 528 nm emis-sion filter set Set the scale value for the automatic sensitivity adjustment to 20,000

5 Readings taken from the parental Vero cells represent ground levels of autofluorescence and are accordingly sub-tracted from the experimental measurements After background subtraction, set the fluorescence value obtained from the unintoxicated (control) Vero-d2EGFP cells arbitrarily at 100% Express all experimental data as percentages of this 100% control value Examples of the results produced are

6 When screening toxin inhibitors, apply each inhibitor to Vero- d2EGFP cells in the absence of toxin to establish a separate control (100%) value for that condition Express readings from Vero-d2EGFP cells incubated with both toxin and inhib-itor as percentages of the corresponding control value from cells treated with the inhibitor alone This procedure corrects for variability that could result from inhibitor autofluorescence and/or inhibitor effects on cell viability in the absence of

3.3 Fluorescence

Measurements

and Data Analysis

Patrick Cherubin et al.